Career Goals: The experiments outlined in this proposal reflect my goal to better understand normal hematopoietic development at a molecular level, while simultaneously making substantive contributions to this understanding. Insofar as malignant hematopoiesis reflects a dysregulated extension of normal hematopoiesis, a molecular understanding will help identify new therapeutic targets for blood disorders. Exploiting these targets represents a longer-term goal for my research career. In keeping with my training as a physician-scientist it is important that I honor the traditions of patient care, teaching, and scholarship as well. Research Project: MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional co-repressors that alter hematopoietic progenitor cell proliferation and lineage allocation. More broadly, MTG proteins are critical regulators of normal cell growth and development and are preferred targets of chromosomal translocations found in acute myeloid leukemia (AML). Preliminary data suggests MTG16 regulates Notch-dependent signaling. Through distinct motifs, MTG16 binds core components of the Notch transcription complex, CSL and the intracellular domains of Notch receptors (N-ICD) 1-4, and augments transactivation of a N-ICD responsive promoter. This effect of MTG16 requires the CSL and N-ICD binding domains. The N-ICD interacting motif in MTG16 lies immediately adjacent to an absolutely conserved serine (S39), phosphorylated in MTG family proteins and a translocation fusion protein with RUNX1. Aspartate substitution at S39 impairs N-ICD binding to MTG16 and abolishes its potentiating effect toward a Notch-responsive promoter element. Furthermore, hematopoietic progenitors from Mtg16 -/- mice display altered hematopoiesis de novo and after Notch pathway activation in vitro. Enforced MTG16 expression in Mtg16 -/- progenitors restores Notch-dependent, ex vivo lymphoid lineage allocation in a manner that requires the N-ICD binding site. We hypothesize that MTG16 regulates Notch-dependent gene expression and hematopoietic cell fate specification by controlling the composition and integrity of transcriptional regulatory complexes at Notch responsive promoters. We also hypothesize that these effects are regulated by upstream signaling kinases. To address these hypotheses, we plan a focused interrogation of the structural and functional relationships between MTG16 and the core elements of canonical Notch signaling, CSL, N-ICD and Mastermind, and a comprehensive kinome screen directed toward S39 (specific aim #1). We will also employ co-culture and mouse transplantation models to define the impact of MTG16 on hematopoietic cell fate specification involving Notch signaling both in vivo and ex vivo (specific aim #2). Career Development Plan/Environment: The research described, as well as a combination of didactic and experiential training constitutes the majority of the career development plan. Vanderbilt provides an exceptionally collaborative and supportive environment in which to achieve these goals. PUBLIC HEALTH RELEVANCE: Disordered blood cell development is a common feature of human disease, both primarily and as a secondary manifestation. Prominent among the primary disorders is acute myeloid leukemia, a common and often fatal cancer of blood precursor cells that afflicts both children and adults. Understanding normal blood cell development and its regulation is essential to develop effective interventions for problems of blood cell development generally, and may disclose new therapeutic targets for cancers of the blood.